Detection of pathogenic bacteria during rhinovirus infection is associated with increased respiratory symptoms and asthma exacerbations Kirsten M. Kloepfer, MD, MS,a Wai Ming Lee, PhD,b Tressa E. Pappas, BS,b Theresa J. Kang, BS,b Rose F. Vrtis, BS,c Michael D. Evans, MS,d Ronald E. Gangnon, PhD,d Yury A. Bochkov, PhD,b Daniel J. Jackson, MD,b,c Robert F. Lemanske, Jr, MD,b,c and James E. Gern, MDb,c Indianapolis, Ind, and Madison, Wis Background: Detection of either viral or bacterial pathogens is associated with wheezing in children; however, the influence of both bacteria and viruses on illness symptoms has not been described. Objective: We evaluated bacterial detection during the peak rhinovirus season in children with and without asthma to determine whether an association exists between bacterial infection and the severity of rhinovirus-induced illnesses. Methods: Three hundred eight children (166 with asthma and 142 without asthma) aged 4 to 12 years provided 5 consecutive weekly nasal samples during September and scored cold and asthma symptoms daily. Viral diagnostics and quantitative PCR for Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis were performed on all nasal samples. Results: Detection rates were 53%, 17%, and 11% for H influenzae, S pneumoniae, and M catarrhalis, respectively, with From athe Department of Pediatrics, Indiana University School of Medicine, Indianapolis, and the Departments of bPediatrics, cMedicine, and dBiostatistics and Medical Informatics, University of Wisconsin–Madison. The following National Institutes of Health (NIH) grants supported this research: U19 AI070503-01 (RhinoGen); P01 HL070831 (Childhood Origins of Asthma); 1UL1RR025011 from the Clinical and Translational Science Award (CTSA) program of the National Center for Research Resources, NIH; and T32AI007635 (University of Wisconsin Allergy Research Training program). Disclosure of potential conflict of interest: K. M. Kloepfer, T. E. Pappas, M. D. Evans, and Y. A. Bochkov have received research support from the National Institutes of Health (NIH). R. E. Gangnon has received research support from the National Heart, Lung, and Blood Institute (NHLBI). D. J. Jackson has received consulting fees from GlaxoSmithKline and Genentech. R. F. Lemanske, Jr, has been supported by a grant, consultancy fees, and participation fees from the NIH; is a board member for the American Academy of Allergy, Asthma & Immunology (AAAAI); has received consultancy fees from Merck, Sepracor, SA Boney and Associates, GlaxoSmithKline, the American Institute of Research, Genentech, Double Helix Development, and Boehringer Ingelheim; is employed by the University of Wisconsin School of Medicine and Public Health; has received research support from the NHLBI and Pharmaxis; has received lecture fees from the Michigan Public Health Institute, Allegheny General Hospital, the American Academy of Pediatrics, West Allegheny Health Systems, California Chapter 4, AAP, the Colorado Allergy Society, the Pennsylvania Allergy and Asthma Association, Harvard Pilgrim Health, the California Society of Allergy, the NYC Allergy Society, the World Allergy Organization, the American College of Chest Physicians, APAPARI, and the Western Society of Allergy, Asthma, and Immunology; has received payment for manuscript preparation from the AAAAI; and has received royalties from Elsevier and UpToDate. J. E. Gern has received research support from the NIH, Merck, AstraZeneca, and GlaxoSmithKline and has received personal fees from GlaxoSmithKline, Biota, Centocor, Boehringer Ingelheim, MedImmune, Theraclone, Merck, and Gilead. The rest of the authors declare that they have no relevant conflicts of interest. Received for publication September 24, 2013; revised January 21, 2014; accepted for publication February 14, 2014. Available online April 3, 2014. Corresponding author: Kirsten M. Kloepfer, MD, MS, 705 Riley Hospital Dr, RI 2606, Indianapolis, IN 46202. E-mail: [email protected]. 0091-6749/$36.00 Ó 2014 American Academy of Allergy, Asthma & Immunology http://dx.doi.org/10.1016/j.jaci.2014.02.030

detection of rhinovirus increasing the risk of detecting bacteria within the same sample (odds ratio [OR], 2.0; 95% CI, 1.4-2.7; P < .0001) or the following week (OR, 1.6; 95% CI, 1.1-2.4; P 5 .02). In the absence of rhinovirus, S pneumoniae was associated with increased cold symptoms (mean, 2.7 [95% CI, 2.0-3.5] vs 1.8 [95% CI, 1.5-2.2]; P 5 .006) and moderate asthma exacerbations (18% [95% CI, 12% to 27%] vs 9.2% [95% CI, 6.7% to 12%]; P 5 .006). In the presence of rhinovirus, S pneumoniae was associated with increased moderate asthma exacerbations (22% [95% CI, 16% to 29%] vs 15% [95% CI, 11% to 20%]; P 5 .01). Furthermore, M catarrhalis detected alongside rhinovirus increased the likelihood of experiencing cold symptoms, asthma symptoms, or both compared with isolated detection of rhinovirus (OR, 2.0 [95% CI, 1.0-4.1]; P 5 .04). Regardless of rhinovirus status, H influenzae was not associated with respiratory symptoms. Conclusion: Rhinovirus infection enhances detection of specific bacterial pathogens in children with and without asthma. Furthermore, these findings suggest that M catarrhalis and S pneumoniae contribute to the severity of respiratory tract illnesses, including asthma exacerbations. (J Allergy Clin Immunol 2014;133:1301-7.) Key words: Rhinovirus, bacteria, asthma

There is mounting evidence that asthma is associated with changes in the airway microbiome. For example, detection of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis within the upper airway in early infancy is associated with an increased risk of recurrent wheezing and then asthma.1,2 In addition, culture-independent methods of bacterial detection demonstrate that Proteobacteria, a phylum of bacteria containing a majority of gram-negative bacteria, appeared more frequently in both nasal and bronchial samples in patients with stable asthma and are linked to increased bronchial hyperresponsiveness.3-5 However, prospective studies have not used molecular methods of detection to analyze these bacteria between children with and without asthma. Recently, we reported that 90% of children with asthma are infected with rhinoviruses during the month of September and that the severity of clinical illness varies from no symptoms to severe wheezing illnesses.6 These findings strongly suggest that there are cofactors that affect the relationship between rhinovirus infection and illness and suggest that airway bacteria might contribute in this regard. Furthermore, a recent study found an increase in invasive pneumococcal disease during peak rhinovirus activity.7 Therefore we hypothesized that detection of specific bacterial pathogens would be increased in children with asthma 1301

1302 KLOEPFER ET AL

Abbreviations used FENO: Fraction of exhaled nitric oxide OR: Odds ratio

compared with children without asthma and that codetection of these bacteria with rhinovirus would be associated with increased respiratory symptoms compared with rhinovirus detection alone. To test these hypotheses, we prospectively obtained weekly nasal secretion specimens from children with and without asthma during the fall of 2007-2009 and then compared detection of viruses and select bacteria with cold and asthma symptoms and moderate asthma exacerbations.

METHODS Study subjects and design Children included in this analysis were enrolled in a larger study (RhinoGen) to determine genetic correlates with more severe rhinovirusinduced illnesses. Of the 383 children participating in RhinoGen, 308 children aged 4 to 12 years submitted samples during a peak rhinovirus season. Starting the first Saturday of September 2007, 2008, or 2009, nasal samples were collected weekly for a total of 5 consecutive weeks. Children, with the help of their parents, were instructed to record upper respiratory tract illnesses and asthma symptoms, morning peak expiratory flow, and albuterol use on daily diary cards from 1 week before the first nasal sample submission through 1 week after the final (fifth) sample submission. See the Methods section in this article’s Online Repository at www.jacionline.org for additional details about recruitment and inclusion criteria. This study was approved by the University of Wisconsin Human Subjects Committee, and written informed consent was obtained from the parents.

J ALLERGY CLIN IMMUNOL MAY 2014

was defined as the 7-day period surrounding (63 days) the day of nasal specimen collection. Each week was designated as positive or negative for virus, bacteria, or both based on viral detection, bacterial detection, or both. Cold and asthma symptom burdens for each week were calculated by summing the daily symptom scores across the week. An illness was defined as a period of 2 or more consecutive days of cold or asthma symptoms that did not contain any periods of consecutive asymptomatic days. All nasal samples obtained between 3 days before the first day of the illness and 3 days after the last day of the illness were considered to be associated with that illness. Cold and asthma symptom burdens for each illness were calculated by summing daily symptom scores across the illness.

Statistical analysis Atopic status (positive determined based on either specific IgE level or skin prick test response), race, and sex were compared by asthma status with the x2 test for association. Age and fraction of exhaled nitric oxide (FENO) were compared by asthma status with the Wilcoxon rank sum test. FEV1, forced vital capacity, and FEV1/forced vital capacity ratio were compared by asthma status with linear models that also included covariates for age, sex, race, and height. Viral infection and bacterial detection rates were compared by asthma status with logistic regression models, both univariate and adjusted for sex, race, and sensitization. Generalized linear mixedeffects models with a random effect for subject to account for repeated measures within subjects were used to compare weekly symptom burdens and occurrences of moderate asthma exacerbations by virus and bacterial detection and to assess whether infection with a given virus or bacterium increased or decreased susceptibility to another bacterium or virus during the same week or 1 week later. A matched-pairs case-control design was used in which each illness (case) was matched with an asymptomatic period (control) of the same length in another subject to assess the association between viral detection, bacterial detection, or both and the occurrence of an illness. Conditional logistic regression was then used to estimate odds ratios (ORs) of illness occurrence with respect to detection of the virus, bacterium, or both. A 2-sided P value of less than .05 was regarded as statistically significant.

Procedures and definitions At the first study visit, subjects were taught to collect samples of their own nasal mucus using a nose-blowing technique, as previously described.6,8 These samples were analyzed for common respiratory tract viruses by using the Respiratory Multicode Assay (EraGen Biosciences, Madison, Wis).9 Nasal samples were also analyzed for S pneumoniae, H influenzae, and M catarrhalis by using quantitative real-time PCR. DNA was extracted with the BiOstic Bacteremia DNA Isolation Kit (Mo Bio laboratories, Carlsbad, Calif). The quantitative Spn9802 PCR for the detection of S pneumoniae10 was combined with the P6 PCR for the detection of H influenzae11 and the copB PCR for the detection of M catarrhalis,12 as previously described. All primers and probes were obtained from Applied Biosystems (Foster City, Calif), and the real-time PCR assay was performed in a 7300 Applied Biosystems instrument. We confirmed double- and triple-positive results obtained by using a Multicode assay by repeating quantitative PCR with single-target assays. Standard curves consisted of bacterial DNA extracted from known quantities of clinical isolates of each bacterium obtained from the University of Wisconsin Hospital Clinical Microbiology Laboratory, and PCR results are expressed as colony-forming unit equivalents. Children scored cold and asthma symptom severity based on a 4-point scoring system (see Table E1 in this article’s Online Repository at www. jacionline.org).6,8 Moderate asthma exacerbations were defined as at least _2) and either a decrease in peak expiratory moderate asthma symptoms (score > flow of at least 20% or increased use of albuterol for 2 days or more in accordance with National Heart, Lung, and Blood Institute and American Thoracic Society definitions.13,14 Current asthma was diagnosed at study completion based on previously reported criteria.15 Skin prick testing and measurement of total and allergen-specific IgE levels in plasma were performed on enrollment (UniCAP 100; Phadia, Uppsala, Sweden). Children who collected at least 4 of 5 weekly nasal specimens and were missing less than 20% of diary card data were included in the analysis. A week

RESULTS Subjects’ characteristics Of the 383 children enrolled in RhinoGen, 54 had insufficient quantity of nasal samples to allow analysis for both viruses and bacteria, whereas an additional 19 subjects did not submit at least 4 (80%) of the 5 scheduled nasal samples, and 2 additional subjects did not complete at least 40 days (80%) of symptom diaries. Therefore a total of 308 (80%) subjects were included in the final analysis (see Fig E1 in this article’s Online Repository at www.jacionline.org). Of the 308 subjects enrolled, 166 (54%) had current asthma. In comparing the groups, there was no significant difference in age or baseline lung function; however, the asthma group had a higher rate of allergic sensitization and higher baseline FENO values and contained more male and fewer white subjects (Table I). Identification of pathogenic bacteria The 308 subjects who completed the study submitted 1394 nasal samples. One hundred seventy-three samples positive for other viruses were excluded from analysis, resulting in 867 (71%) samples positive for bacteria and 481 (39%) samples positive for rhinovirus. Of the bacteria detected, H influenzae was detected most frequently (53% of samples), followed by S pneumoniae (17%) and M catarrhalis (11%). Detection rates were similar between children, regardless of asthma (Table II) and allergic sensitization status (data not shown).

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TABLE I. Study subjects

Age (y) Sex (male) Race (white) Atopic status FENO FEV1 FVC FEV1/FVC ratio

TABLE III. Influence of virus on bacterial detection

Asthma (n 5 166)

No asthma (n 5 142)

P value

8.3 6 2.1 67% 81% 73% 23 6 22 1.71 6 0.44 2.12 6 0.59 0.81 6 0.07

8.6 6 1.6 56% 92% 53% 13 6 13 1.76 6 0.45 2.14 6 0.59 0.83 6 0.07

.27 .03 .007 .0003